Peptide-mediated non-covalent delivery of active agents across the blood-brain barrier

ABSTRACT

The peptides described herein can function as carrier peptides. These peptides can associate with (e.g., non-covalently bind) biologically active molecules or imaging agents to transport the biologically active molecules or imaging across the blood-brain barrier. In some cases, such transport may increase the effectiveness of the biological molecules or imaging agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/225,412, filed on Jul. 14, 2009, which is incorporated byreference in its entirety herein.

TECHNICAL FIELD

This disclosure relates to carrier peptides, including compositions andmethods of using the same. In particular, the disclosure relates tocarrier peptides capable of delivering active agents across theblood-brain barrier and compositions and methods of using the same.

BACKGROUND

The blood-brain barrier (BBB) prevents most macromolecules (e.g., DNA,RNA, and polypeptides) and many small molecules from entering the brain.The BBB is principally composed of specialized endothelial cells withhighly restrictive tight junctions, consequently, passage of substances,small and large, from the blood into the central nervous system iscontrolled by the BBB. This structure makes treatment and management ofpatients with neurological diseases and disorders (e.g., brain cancerand Alzheimer's disease) difficult as many therapeutic agents cannot bedelivered across the BBB with desirable efficiency.

SUMMARY

The peptides described herein can function as carrier peptides. Thesepeptides can associate with (e.g., non-covalently bind) biologicallyactive molecules and imaging agents to transport the biologically activemolecules and imaging agents across the blood-brain barrier. In somecases, such transport may increase the effectiveness of the biologicalmolecules and imaging agents.

A carrier peptide, as described herein, can include the sequence:

X_(n)-[B]_(m)

or a pharmaceutically acceptable salt thereof, wherein:X is a hydrophilic amino acid;B is a blood-brain barrier agent;n is an integer from 4 to 50; andm is integer from 1 to 3.

In some embodiments, the blood-brain barrier agent is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R.

A hydrophilic amino acid (X) can be chosen from arginine, asparagine,aspartic acid, glutamic acid, glutamine, histidine, lysine, serine,threonine, tyrosine, or a combination thereof. For example, in someembodiments, X_(n) can comprise ten lysines and six arginines; sixlysines and sixteen arginines; eight lysines, eight arginines, and eighthistidines. In some embodiments, X is lysine.

The variable n is an integer ranging from 4 to 50 (e.g., 4, 6, 8, 10,12, 16, 20, 24, 26, 28, 32, 36, 40, 42, 44, 48, and 50). In someembodiments, n is chosen from 4, 8, 12, 16, and 20. In some embodiments,n is 16. In some embodiments, m is 1.

The blood-brain barrier agent can be a receptor binding domain of anapolipoprotein. For example, the receptor binding domain of anapolipoprotein can be chosen from the receptor binding domain of ApoA,ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3, and ApoE4. In some embodiments,the receptor binding domain of an apolipoprotein is chosen from thereceptor binding domain of ApoB and ApoE.

The blood-brain barrier agent can also include a polypeptide sequencehaving at least 80% sequence identity to:

(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 14)S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 15)Y-P-A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y-L-N-L-V-T-R-Q-R-Y*; (SEQ ID NO: 16)A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R- H-Y-L-N-L-V-T-R-Q-R-Y*;(SEQ ID NO: 17) Y-P-S-D-P-D-N-P-G-E-D-A-P-A-E-D-L-A-R-Y-Y-S-A-L-R-H-Y-I-N-L-I-T-R-Q-R-Y*; or (SEQ ID NO: 18)A-P-L-E-P-V-Y-P-G-D-D-A-T-P-E-Q-M-A-Q-Y-A-A-E-L-R-R-Y-I-N-M-L-T-R-P-R-Y*,wherein Y* is tyrosine or a tyrosine derivative. In some embodiments,the polypeptide is less than 100 amino acids in length.

Examples of a carrier peptide can include:

(SEQ ID NO: 19) K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A;(SEQ ID NO: 20) K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L- R-K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 24)K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 25)K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 26)K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V- E-G-S-H;(SEQ ID NO: 27) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S- N-K-F-V-E-G-S-H; and(SEQ ID NO: 28) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H.

Further provided herein is a complex, or a pharmaceutically acceptablesalt thereof, having a biologically active molecule or an imaging agentassociated with a carrier peptide, as described above. In someembodiments, the biologically active molecule or imaging agent isnon-covalently bound to the carrier peptide.

An imaging agent can be any chemical or substance which is used toprovide the signal or contrast in imaging. For example, the imagingagent can be ¹²⁵I-IgG or magnevist. A biologically active molecule caninclude a polypeptide; oligonucleotide; plasmid; small molecule;antibody; antibody fragment; carbohydrate; polysaccharide; lipid;glycolipid; antigen; and antigenic peptide. In some embodiments, thebiologically active molecule is chosen from a: polypeptide;oligonucleotide; and plasmid. An oligonucleotide can include a codingDNA sequence; antisense DNA sequence; mRNA, antisense RNA sequence;RNAi; and siRNA. In some embodiments, the biologically active moleculeis a small molecule, for example, a therapeutic agent.

The carrier peptides and complexes of carrier peptides and biologicallyactive agents or imaging agents have various uses and can be used invarious methods. For example, provided herein is a method oftransporting a biologically active molecule or imaging agent across theblood-brain barrier of a subject. The method can include administeringto the subject a complex, or a pharmaceutically acceptable salt thereof,having the biologically active molecule or imaging agent associated witha carrier peptide.

Also provided is a method of treating a brain disorder in a subject. Themethod can include administering to the subject a complex, or apharmaceutically acceptable salt thereof, having a biologically activeagent associated with a carrier peptide. The brain disorder can bechosen from meningitis, epilepsy, multiple sclerosis, neuromyelitisoptica, late-stage neurological trypanosomiasis, Parkinson's,progressive multifocal leukoencephalopathy, De Vivo disease, Alzheimer'sdisease, HIV Encephalitis, and cancer.

This disclosure further provides a method of imaging the central nervoussystem of a subject. The method can include administering to the subjecta complex, or a pharmaceutically acceptable salt thereof, comprising animaging agent associated with a carrier peptide; and imaging the centralnervous system of the subject.

Pharmaceutical compositions are also disclosed. A pharmaceuticalcomposition can include a complex, or a pharmaceutically acceptable saltthereof, having a biologically active molecule or imaging agent, acarrier peptide, and a pharmaceutically acceptable carrier.

Finally, kits are also provided. A kit can include a carrier peptide. Insome embodiments, the kit further includes a biologically activemolecule or an imaging agent.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a series of images illustrating the differences betweendelivery of Alexa-IgG complexed to K₁₆ApoE into brain and uncomplexedAlexa-IgG.

FIG. 2 illustrates the accumulation of radioactive IgG 4.1 in mousebrain aided by K₁₆ApoE.

FIG. 3 shows delivery of K16APoE-mediated beta-galactosidase in brain(Top) and in other organs (Bottom). In one experiment,beta-galactosidase was injected in mice mixed with (FIG. 3A′) andwithout K16ApoE (FIG. 3A), brain slices made six hours after injectionwere stained for enzyme activity (top). In a separate relatedexperiment, slides were made from different organs and stained forbeta-galactosidase activity after delivery without (FIG. 3B-G) and withK16ApoE (FIG. 3B′-G′), B—brain; C—heart, D—kidney, E—lung, F—liver,G—spleen.

FIG. 4 shows the evaluation of K16ApoE-mediated delivery of IgG (A) andIgM (B) in mice brains by microSPECT. Imaging was done at 1 hourintervals up to six hours, after which cardiac perfusion was performed,and final imaging carried out 30 minutes after perfusion. Left bars—withK16ApoE; Right bars—no K16ApoE.

FIG. 5 illustrates labeling of amyloid plaques with a plaque-specificantibody delivered via K16ApoE (SEQ ID NO: 22) in brains of mice modelsof Alzheimer's disease. Two mice with Alzheimer's disease were used: onerepresented by A, C and E, while the other is represented by B, D and F.A, C and E represent adjacent brain sections from one mouse, whereas B,D and F represent adjacent brain sections from another mouse. A,B—thioflavine S staining; C, D—immunostaining to identify plaques usingthe 4G8 as the primary antibody, and an anti-mouse antibody as thesecondary antibody; E, F—immunostaining using the secondary antibodyonly. The 4G8 IgG was injected in the first mouse (left panel) withoutK16ApoE, while the second mouse (right panel) received injection of theIgG mixed with K16ApoE.

FIG. 6 shows the evaluation of the integrity of the BBB after injectionof K16ApoE (SEQ ID NO: 22). Each mouse (represented by A through G)received an injection of K16ApoE, followed by injection ofβ-galactosidase at the indicated times. Brain slices were made 4 hoursafter β-galactosidase injection and were followed by staining foractivity of the enzyme. A—β-galactosidase was injected 1 minute afterK16ApoE injection. B—β-galactosidase was injected 5 minutes afterK16ApoE injection. C—β-galactosidase was injected 10 minutes afterK16ApoE injection. D—β-galactosidase was injected 30 minutes afterK16ApoE injection. E—β-galactosidase was injected 1 hour after K16ApoEinjection. F—β-galactosidase was injected 2 hours after K16ApoEinjection. G—β-galactosidase was injected 4 hours after K16ApoEinjection. H—positive control (β-galactosidase mixed with K16ApoE wasinjected, brain slices were made 4 hours after the injection andproceeded for staining for enzyme activity).

FIG. 7 demonstrates the need for chemical linking of the K16 and theApoE peptide moieties for non-covalent delivery across the BBB. Deliveryof β-galactosidase in brain with K16 peptide alone (A), with ApoEpeptide only (B), with K16 peptide+ApoE peptide (C), and with K16APoEpeptide (D).

FIG. 8 illustrates the testing mutant ApoE peptides for their potentialas non-covalent transporters of proteins across the BBB. A—no peptide,no β-gal; B—K16ApoE+β-gal; C—K16L3N7+β-gal; D—K16L3L20+β-gal;E—K16N7L20+β-gal.

FIG. 9 shows a time course study of β-galactosidase delivery in mousebrain with K16ApoE. A—13-galactosidase, no peptide, 6 h;B—β-galactosidase+peptide, 1 h; C—β-galactosidase+peptide, 2 h;D-β-galactosidase+peptide, 5 h; E-β-galactosidase+peptide, 10 h.

DETAILED DESCRIPTION

The peptides described herein can function as carrier peptides. Thesepeptides can associate with (e.g., non-covalently bind) biologicallyactive molecules and imaging agents to transport the biologically activemolecules and imaging agents across the blood-brain barrier. In somecases, such transport may increase the effectiveness of the biologicalmolecules and imaging agents.

Carrier Peptides

Provided herein are carrier peptides having the following sequence:

X_(n)-[B]_(m)

or a pharmaceutically acceptable salt, thereof, wherein:X is a hydrophilic amino acid;B is a blood-brain barrier agent;n is an integer from 4 to 50; andm is integer from 1 to 3.

In some embodiments, the blood-brain barrier is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R.

A hydrophilic amino acid can be chosen from: arginine, asparagine,aspartic acid, glutamic acid, glutamine, histidine, lysine, serine,threonine, tyrosine, and combinations and non-natural derivativesthereof. In some embodiments, a hydrophilic amino acid can be chosenfrom lysine or a non-natural lysine derivative, arginine or anon-natural arginine derivative, and combinations thereof. In someembodiments, the hydrophilic amino acid is lysine. Non-limiting examplesof X_(n) can include KKKK (SEQ ID NO:1); KKKKKKKK (SEQ ID NO:2);KKKKKKKKKKKK (SEQ ID NO:3); KKKKKKKKKKKKKKKK (SEQ ID NO:4); RRRR (SEQ IDNO:5); RRRRRRRR (SEQ ID NO:6); RRRRRRRRRRRR (SEQ ID NO:7);RRRRRRRRRRRRRRRR (SEQ ID NO:8); KRKR (SEQ ID NO:9); KKKR (SEQ ID NO:10);KKKRRRKKKRRR (SEQ ID NO:11); and KKKKRRRRKKKKRRRR (SEQ ID NO:12).

The variable n is an integer ranging from 4 to 50 (e.g., 4, 6, 8, 10,12, 16, 20, 24, 26, 28, 32, 36, 40, 42, 44, 48, and 50). For example, ncan range from 4 to 20. In some embodiments, n is chosen from 4, 8, 12,16, and 20. For example, n can be 16. In some embodiments, m is 1.

A blood-brain barrier agent, as used herein, is any polypeptide ornon-polypeptide ligand that can cross the blood-brain barrier. In someembodiments, a blood-brain barrier agent has a cognate receptor on braincells or can bind to such receptors. In some embodiments, theblood-brain barrier agent comprises a transferring-receptor binding siteof a transferrin. In some embodiments, the blood-brain barrier agentcomprises a receptor binding domain of an apolipoprotein. A receptorbinding domain of an apolipoprotein, for example, can be chosen from thereceptor binding domain of ApoA, ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3,ApoE4, and combinations thereof. In some embodiments, the receptorbinding domain of an apoliprotein is chosen from the receptor bindingdomain of ApoB and ApoE.

In some embodiments, the blood-brain barrier agent comprises a sequencehaving at least 80% (e.g., at least 85%; at least 90%; at least 92%; atleast 95%; at least 98%; and at least 99%) sequence identity to:

(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 14)S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 15)Y-P-A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y-L-N-L-V-T-R-Q-R-Y*; (SEQ ID NO: 16)A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R- H-Y-L-N-L-V-T-R-Q-R-Y*;(SEQ ID NO: 17) Y-P-S-D-P-D-N-P-G-E-D-A-P-A-E-D-L-A-R-Y-Y-S-A-L-R-H-Y-I-N-L-I-T-R-Q-R-Y*; or (SEQ ID NO: 18)A-P-L-E-P-V-Y-P-G-D-D-A-T-P-E-Q-M-A-Q-Y-A-A-E-L-R-R-Y-I-N-M-L-T-R-P-R-Y*,wherein Y* is tyrosine or a tyrosine derivative (e.g., an amidatedtyrosine). See, e.g., Ballantyne, G. H., Obesity Surgery, 16:651-6582006.

In some embodiments, the blood-brain barrier agent includes apolypeptide comprising the following sequence:

(SEQ ID NO: 29) L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8-R-D-X9wherein:X1 is selected from the group consisting of A, L, S, and V;X2 is selected from the group consisting of L and M;X3 is selected from the group consisting of A and S;X4 is selected from the group consisting of N, S, and T;X5 is selected from the group consisting of K and N;X6 is selected from the group consisting of L, M, and V;X7 is selected from the group consisting of R and P;X8 is selected from the group consisting of L and M; andX9 is selected from the group consisting of A and L.Non-limiting examples of a blood-brain barrier agents according to thissequence include:

(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 30)L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 31)L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 32)L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 33)L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 34)L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 35)L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 36)L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 37)L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 38)L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 39)L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 40)L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 41)L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 42)L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 43)L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 44)L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 45)L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 129)L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; and (SEQ ID NO: 130)L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.

In some embodiments, the blood-brain barrier agent is less than 100amino acids in length (e.g., less than 90 amino acids in length; lessthan 80 amino acids in length; less than 75 amino acids in length; lessthan 70 amino acids in length; less than 65 amino acids in length; lessthan 62 amino acids in length; less than 60 amino acids in length; lessthan 55 amino acids in length; less than 50 amino acids in length; andless than 45 amino acids in length).

“Percent sequence identity” refers to the degree of sequence identitybetween any given reference sequence, e.g., SEQ ID NO:13, and acandidate blood-brain barrier agent sequence. A candidate sequencetypically has a length that is from 80 percent to 200 percent of thelength of the reference sequence (e.g., 82, 85, 87, 89, 90, 93, 95, 97,99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200percent of the length of the reference sequence). A percent identity forany candidate nucleic acid or polypeptide relative to a referencenucleic acid or polypeptide can be determined as follows. A referencesequence (e.g., a nucleic acid sequence or an amino acid sequence) isaligned to one or more candidate sequences using the computer programClustalW (version 1.83, default parameters), which allows alignments ofnucleic acid or polypeptide sequences to be carried out across theirentire length (global alignment). Chema et al., Nucleic Acids Res.,31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of peptide sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of peptidesequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.; gap extension penalty: 0.5; hydrophilic gaps: on;hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys;residue-specific gap penalties: on. The ClustalW output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launchersite (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and atthe European Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

The variable m is an integer from 1 to 3. In some embodiments, m is 1.

In some embodiments, a carrier peptide can be chosen from:

(SEQ ID NO: 19) K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A;(SEQ ID NO: 20) K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L- R-K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 24)K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 25)K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 26)K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V- E-G-S-H;(SEQ ID NO: 27) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S- N-K-F-V-E-G-S-H; and(SEQ ID NO: 28) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H.

In some embodiments, a carrier peptide can be chosen from:

[X]_(n)-L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8- R-D-X9wherein:X is a hydrophilic amino acid;n is an integer from 4 to 20;X1 is selected from the group consisting of A, L, S, and V;X2 is selected from the group consisting of L and M;X3 is selected from the group consisting of A and S;X4 is selected from the group consisting of N, S, and T;X5 is selected from the group consisting of K and N;X6 is selected from the group consisting of L, M, and V;X7 is selected from the group consisting of R and P;X8 is selected from the group consisting of L and M; andX9 is selected from the group consisting of A and L.Non-limiting examples of a blood-brain barrier agents according to thissequence include:

(SEQ ID NO: 19) K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A;(SEQ ID NO: 20) K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L- R-K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 46)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 47)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 48) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 49)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 50)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 51)K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 52)K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 53) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 54)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 55)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 56)K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 57)K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 58) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 59)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 60)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 61)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 62)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L- L-R-D-A;(SEQ ID NO: 63) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 64)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 65)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 66)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 67)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L- M-R-D-A;(SEQ ID NO: 68) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 69)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 70)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 71)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 72)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 73) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 74)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 75)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 76)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 77)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L- L-R-D-A;(SEQ ID NO: 78) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 79)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 80)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 81)K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 82)K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 83) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 84)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 85)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 86)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 87)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L- L-R-D-A;(SEQ ID NO: 88) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 89)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 90)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 91)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 92)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L- L-R-D-A;(SEQ ID NO: 93) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 94)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 95)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 96)K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 97)K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 98) K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 99)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 100)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 101)K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 102)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L- M-R-D-A;(SEQ ID NO: 103) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 104)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 105)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 106)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 107)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L- M-R-D-A;(SEQ ID NO: 108) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 109)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 110)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 111)K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 112)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 113) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 114)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 115)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 116)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 117)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L- M-R-D-A;(SEQ ID NO: 118) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 119)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 120)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 121)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 122)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L- L-R-D-A;(SEQ ID NO: 123) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 124)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 125)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 131)K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 132)K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L- L-R-D-L;(SEQ ID NO: 133) K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 134)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 135)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 136)K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 137)K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L- L-R-D-L ;(SEQ ID NO: 138) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 139)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 140)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.

Complexes

Also provided herein are complexes having a biologically active moleculeor an imaging agent associated with a carrier peptide as describedherein. In some embodiments, the biologically active molecule or imagingagent is non-covalently bound to the carrier peptide.

As used herein, a “biologically active molecule” includes any moleculewhich, if transported across the blood-brain barrier, can have abiological effect. Examples of biologically active molecules includepolypeptides, which include functional domains of biologically activemolecules, particular examples include growth factors, enzymes,transcription factors, toxins, antigenic peptides (as for vaccines),antibodies, and antibody fragments. For example, brain derivedneurotrophic factor, fibroblast growth factor (e.g., (FGF)-2 or multipleFGFs), nerve growth factor, neurotrophin (e.g., NT-3 and NT-4/5), glialderived neurotrophic factor, ciliary neurotrophic factor, neurturin,neuregulins, interleukins, transforming growth factor (e.g., TGF-α andTGF-β), vasoactibe intestinal peptide, epidermal growth factor (EGF),erythropoietin, heptocytel growth factor, platelet derived growthfactor, artemin, persephin, netrins, cardiotrophin-1, stem cell factor,midkine, pleiotrophin, bone morphogenic proteins, saposins, semaporins,leukemia inhibitory factor, anti-Aβ, anti-HER2, anti-EGF, anti-nogo A,anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand),anti-α-synuclein, anti-htt, anti-prion, anti-West Nile virus,αL-iduronidase, iduronate-2-sulfatase,N-acetyl-galactosamine-6-sulfatase, arylsulfatase B, acid α-glucosidase,and acid sphingomyelinase (See, Pardridge, W. M., Bioconjug. Chem.19(7): 1327-38 2008). Additional examples of biologically activemolecules include oligonucleotides, such as natural or engineeredplasmids, coding DNA sequences, antisense DNA sequences, mRNAs,antisense RNA sequences, RNAis, and siRNAs; carbohydrates; lipids; andglycolipids.

Further examples of biologically active molecules include smallmolecules, including therapeutic agents, in particular those with lowblood-brain barrier permeability. Some examples of these therapeuticagents include cancer drugs, such as daunorubicin and toxic chemicalswhich, because of the lower dosage that can be administered by thismethod, can now be more safely administered. For example, a therapeuticagent can include bevacizumab, irinotecan, zoledronate, andtemozolomide.

In another embodiment, the therapeutic agent can include abroad-spectrum antibiotic (e.g., cefotaxime, ceftriaxone, ampicillin andvancomycin); an antiviral agent (e.g., acyclovir); acetazolamide;carbamazepine; clonazepam; clorazepate dipotassium; diazepam; divalproexsodium; ethosuximide; felbamate; fosphenytoin sodium; gabapentin;lamotrigine; levetiracetam; lorazepam; oxcarbazepine; phenobarbital;phenyloin; phenyloin sodium; pregabalin; primidone; tiagabinehydrochloride; topiramate; trimethadione; valproic acid; zonisamide;copaxone; tysabri; novantrone; donezepil HCL; rivastigmine; galantamine;memantine; levodopa; carbidopa; parlodel, permax, requip, mirapex;symmetrel; artane; cogentin; eldepryl; and deprenyl.

Numerous other examples of biologically active molecules will beapparent to the skilled artisan.

Yet another example of a biologically active molecule is an antigenicpeptide. Antigenic peptides can be administered to provide immunologicalprotection when imported by cells involved in the immune response. Otherexamples include immunosuppressive peptides (e.g., peptides that blockautoreactive T cells, which peptides are known in the art).

Polypeptides from a few amino acids to about a thousand amino acids canbe used. In some embodiments, the size range for polypeptides is from afew amino acids to about 250 amino acids (e.g., about 3 to about 250amino acids; about 20 to about 250 amino acids; about 50 to about 250amino acids; about 100 to about 250 amino acids; about 150 to about 250amino acids; about 3 amino acids to about 200 amino acids; about 3 aminoacids to about 150 amino acids; about 3 amino acids to about 175 aminoacids; about 3 amino acids to about 125 amino acids; about 25 aminoacids to about 200 amino acids; about 50 amino acids to about 150 aminoacids; and about 75 amino acids to about 225 amino acids). For anymolecule, size ranges can be up to about a molecular weight of about 1million. In some embodiments, the size ranges up to a molecular weightof about 25,000, and in particular embodiments, the size ranges can beup to a molecular weight of about 3,000.

By “antisense” it is meant a non-enzymatic nucleic acid molecule thatbinds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (proteinnucleic acid; Egholm et al., 1993 Nature 365, 566) interactions andalters the activity of the target RNA (for a review, see Stein andCheng, 1993 Science 261, 1004; Agrawal et al., U.S. Pat. No. 5,591,721;Agrawal, U.S. Pat. No. 5,652,356). Typically, antisense molecules willbe complementary to a target sequence along a single contiguous sequenceof the antisense molecule. However, in certain embodiments, an antisensemolecule may bind to a substrate such that the substrate molecule formsa loop, and/or an antisense molecule may bind such that the antisensemolecule forms a loop. Thus, the antisense molecule may be complementaryto two (or even more) non-contiguous substrate sequences or two (or evenmore) non-contiguous sequence portions of an antisense molecule may becomplementary to a target sequence or both.

RNA interference (RNAi) and short intervening RNA (siRNA) sequences canbe used to modulate (e.g., inhibit) gene expression (see, e.g., Elbashiret al., 2001, Nature, 411, 494 498; and Bass, 2001, Nature, 411, 428429; Bass, 2001, Nature, 411, 428 429; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914). In one embodiment, a siRNA moleculecomprises a double stranded RNA wherein one strand of the RNA iscomplimentary to the RNA of interest. In another embodiment, a siRNAmolecule comprises a double stranded RNA wherein one strand of the RNAcomprises a portion of a sequence of an RNA of interest. In yet anotherembodiment, a siRNA molecule of the invention comprises a doublestranded RNA wherein both strands of RNA are connected by anon-nucleotide linker Alternately, a siRNA molecule of the inventioncomprises a double stranded RNA wherein both strands of RNA areconnected by a nucleotide linker, such as a loop or stem loop structure.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules (i.e.,molecules that contain an antigen binding site that specifically bindsto a peptide). An antibody can be a monoclonal antibody, a polyclonalantibody, a humanized antibody, a fully human antibody, a single chainantibody, a chimeric antibody, or a fragment thereof. The term “antibodyfragment” of a full length antibody refers to one or more fragments of afull-length antibody that retain the ability to specifically bind to atarget of interest.

An imaging agent, as used herein, can be any chemical or substance whichis used to provide the signal or contrast in imaging. The signalenhancing domain can be an organic molecule, metal ion, salt or chelate,particle (particularly iron particle), or labeled peptide, protein,polymer or liposome.

In some embodiments, the imaging agent is a physiologically compatiblemetal chelate compound consisting of one or more cyclic or acyclicorganic chelating agents complexed to one or more metal ions with atomicnumbers 21-29, 42, 44, or 57-83.

For x-ray imaging, the imaging agent may consist of iodinated organicmolecules or chelates of heavy metal ions of atomic numbers 57 to 83. Insome embodiments, the imaging agent is ¹²⁵I-IgG. Examples of suitablecompounds are described in M. Sovak, ed., “Radiocontrast Agents,”Springer-Verlag, pp. 23-125 (1984) and U.S. Pat. No. 4,647,447.

For ultrasound imaging, the imaging agent can consist of gas-filledbubbles such as Albunex, Echovist, or Levovist, or particles or metalchelates where the metal ions have atomic numbers 21-29, 42, 44 or57-83. Examples of suitable compounds are described in Tyler et al.,Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, “EnhancementAgents for Ultrasound: Fundamentals,” Pharmaceuticals in MedicalImaging, pp. 682-87. (1990).

For nuclear radiopharmaceutical imaging or radiotherapy, the imagingagent can consist of a radioactive molecule. In some embodiments, thechelates of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In, and Ga can be used. Insome embodiments, the chelates of Tc-99m can be used. Examples ofsuitable compounds are described in Rayudu G V S, Radiotracers forMedical Applications, I, pp. 201 and D. P. Swanson et al., ed.,Pharmaceuticals in Medical Imaging, pp. 279-644 (1990).

For ultraviolet/visible/infrared light imaging, the imaging agent canconsist of any organic or inorganic dye or any metal chelate.

For MRI, the imaging agent can consist of a metal-ligand complex of aparamagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or57-83. In some embodiments, the paramagnetic metal is chosen from:Gd(III), Fe(III), Mn(II and III), Cr(III), Cu(II), Dy(III), Tb(III),Ho(III), Er(III) and Eu(III). Many suitable chelating ligands for MRIagents are known in the art. These can also be used for metal chelatesfor other forms of biological imaging. For example, an imaging agent caninclude:

“Associated with”, as used herein, is meant that the biologically activemolecule or imaging agent is conjugated to the carrier peptide in such amanner that when the carrier peptide crosses the blood-brain barrier,the molecule or agent is also imported. In certain embodiments, thebiologically active molecule or imaging agent is non-covalently bound tothe carrier peptide. For example, the biologically active molecule andthe carrier peptide may be associated through electrostaticinteractions. In other embodiments, the carrier peptide may becovalently bound, either directly or indirectly (e.g., through alinker), to the biologically active molecule or imaging agent.

A linker can be any moiety suitable for linking a carrier peptide to abiologically active molecule. A linker can be bound at the C-terminus,the N-terminus, or both, of a carrier peptide. Additionally, a linkercan be bound to the side chain of a carrier peptide. If a carrierpeptide is bound to multiple linkers, each linker can be different. Alinker can be covalently linked to a side chain of an amino acid, e.g.,lysine, glutamine, cysteine, methionine, glutamate, aspartate,asparagine.

In some embodiments an amino acid side chain can serve as the linker.For example the epsilon amino group (ε-NH₂) can be used to conjugate toa carrier for instance through an amide or thiourea linkage. Similarlythe delta amino group of ornithine (orn), the gamma amino group ofdiaminobutyric acid (dab), or the beta amino group of diamino proprionicacid (dpr) can also act as linkers. These amino acids may be at the C-or N-terminus of the carrier peptide or they may be positioned withinthe carrier peptide sequence.

The complex composed of a biologically active molecule or imaging agentand a carrier peptide can be prepared by any method known by thosehaving ordinary skill in the art. In some embodiments, the carrierpeptide and the biologically active molecule or imaging agent arecombined, incubated at room temperature, and then used. For example,solutions can be prepared of the delivery peptide (K16ApoE) and IgG inPBS (phosphate buffered saline) or OptiMem (commercially available) orcell culture media without serum in desired concentrations. The deliverypeptide and IgG can be mixed in the desired ratios and then incubated atroom temperature for 30-60 minute. Following incubation, the mixture isready for injection/delivery.

Combinations of the biologically active molecule and the carrier peptidecan be prepared at a variety of molar ratios. For example, a molar ratioof biologically active molecule to carrier peptide can range from about1:1 to about 1:200 (e.g., 1:2; 1:5; 1:8; 1:10; 1:25; 1:30; 1:40; 1:45;1:50; 1:60; 1:65; 1:70; 1:75; 1:80; 1:90; 1:100; 1:125; 1:135; 1:145;1:150; 1:175; and 1:190). In some embodiments, a molar excess of carrierpeptide to biologically active molecule is used. For example, the molarratio of biologically active molecule to carrier peptide can range fromabout 1:20 to about 1:100 (e.g, 1:50 to about 1:90). In some cases, theratio can be 1:70.

The term “pharmaceutically-acceptable salt” refers to salts whichpossess toxicity profiles within a range that affords utility inpharmaceutical applications. Pharmaceutically unacceptable salts maynonetheless possess properties such as high crystallinity, which mayrender them useful, for example in processes of synthesis, purificationor formulation of compounds described herein. In general the usefulproperties of the compounds described herein do not depend critically onwhether the compound is or is not in a salt form, so unless clearlyindicated otherwise (such as specifying that the compound should be in“free base” or “free acid” form), reference in the specification to acarrier peptide or a complex comprising a carrier peptide should beunderstood as encompassing salt forms of the compound, whether or notthis is explicitly stated.

Suitable pharmaceutically-acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric and galacturonic acid. Examples ofpharmaceutically unacceptable acid addition salts include, for example,perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts include, forexample, metallic salts including alkali metal, alkaline earth metal andtransition metal salts such as, for example, calcium, magnesium,potassium, sodium and zinc salts. Pharmaceutically acceptable baseaddition salts also include organic salts made from basic amines suchas, for example, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Examples of pharmaceutically unacceptable base addition saltsinclude lithium salts and cyanate salts.

All of these salts may be prepared by conventional means from thecorresponding carrier peptide or complex by reacting, for example, theappropriate acid or base with a carrier peptide or complex as describedherein. Preferably the salts are in crystalline form, and preferablyprepared by crystallization of the salt from a suitable solvent. Aperson skilled in the art will know how to prepare and select suitablesalt forms for example, as described in Handbook of PharmaceuticalSalts: Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth(Wiley-VCH 2002).

Methods of Use

Also provided herein are methods of using carrier peptides to transporta biologically active molecule or imaging agent across the blood-brainbarrier of a subject. The method can include administering to thesubject a complex having a biologically active molecule or imaging agentassociated with a carrier peptide, or a pharmaceutically acceptablesalt, as described herein.

A subject can include both mammals and non-mammals. Mammals include, forexample, humans; nonhuman primates, e.g. apes and monkeys; cattle;horses; sheep; rats; mice; pigs; and goats. Non-mammals include, forexample, fish and birds.

Transporting a biologically active molecule can include importing themolecule across the blood-brain barrier.

Further provided herein is a method of treating a brain disorder in asubject. The method can include administering to the subject a complexcomprising a biologically active agent associated with a carrierpeptide, as described herein. In some embodiments, the biologicallyactive molecule is a therapeutic agent. In some embodiments, the braindisorder is chosen from: meningitis, epilepsy, multiple sclerosis,neuromyelitis optica, late-stage neurological trypanosomiasis,Parkinson's, progressive multifocal leukoencephalopathy, De Vivodisease, Alzheimer's disease, HIV Encephalitis, and cancer.

In some embodiments, the brain disorder is a brain cancer, for exampleastrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma),glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,and congenital tumors; or a cancer of the spinal cord, e.g.,neurofibroma, meningioma, glioma, and sarcoma.

This disclosure also provides a method of imaging the central nervoussystem of a subject. In some embodiments, the method can includeadministering to the subject a complex comprising an imaging agentassociated with a carrier peptide, as described herein, and imaging thecentral nervous system of the subject.

The complex can be administered by any route, e.g., IV, intramuscular,SC, oral, intranasal, inhalation, transdermal, and parenteral.

The complex can be formulated with a pharmaceutically acceptable carrierselected on the basis of the selected route of administration andstandard pharmaceutical practice. The complex may be formulated intodosage forms according to standard practices in the field ofpharmaceutical preparations. See Alphonso Gennaro, ed., Remington'sPharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co.,Easton, Pa. Suitable dosage forms may comprise, for example, tablets,capsules, solutions, parenteral solutions, troches, suppositories, orsuspensions.

For parenteral administration, the complex may be mixed with a suitablecarrier or diluent such as water, an oil (particularly a vegetable oil),ethanol, saline solution, aqueous dextrose (glucose) and related sugarsolutions, glycerol, or a glycol such as propylene glycol orpolyethylene glycol. Solutions for parenteral administration preferablycontain a water soluble salt of the complex. Stabilizing agents,antioxidant agents and preservatives may also be added. Suitableantioxidant agents include sulfite, ascorbic acid, citric acid and itssalts, and sodium EDTA. Suitable preservatives include benzalkoniumchloride, methyl- or propyl-paraben, and chlorbutanol. The compositionfor parenteral administration may take the form of an aqueous ornon-aqueous solution, dispersion, suspension or emulsion.

For oral administration, the complex may be combined with one or moresolid inactive ingredients for the preparation of tablets, capsules,pills, powders, granules or other suitable oral dosage forms. Forexample, the complex may be combined with at least one excipient such asfillers, binders, humectants, disintegrating agents, solution retarders,absorption accelerators, wetting agents absorbents or lubricatingagents.

The specific dose of a complex will, of course, be determined by theparticular circumstances of the individual patient including the size,weight, age and sex of the patient, the nature and stage of the diseasebeing treated, the aggressiveness of the disease disorder, and the routeof administration of the compound.

Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising abiologically active molecule or imaging agent associated with a carrierpeptide. In some embodiments, the biologically active molecule orimaging agent is non-covalently bound to the carrier peptide.

The pharmaceutical compositions provided herein contain a biologicallyactive molecule or imaging agent associated with a carrier peptide in anamount that results in transportation of the biologically activemolecule or imaging agent across the blood-brain barrier, and apharmaceutically acceptable carrier. Pharmaceutical carriers suitablefor administration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

The compositions can be, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration and intraperitonealinjection, as well as transdermal patch preparation, dry powderinhalers, and ointments (see, e.g., Ansel Introduction to PharmaceuticalDosage Forms, Fourth Edition 1985, 126).

The concentration of the biologically active molecule or imaging agentassociated with a carrier peptide in the pharmaceutical composition willdepend on absorption, inactivation and excretion rates of the compounds,the physicochemical characteristics of the compounds, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

The pharmaceutical composition may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

Dosage forms or compositions containing a biologically active moleculeor imaging agent associated with a carrier peptide in the range of0.005% to 100% with the balance made up with a non-toxic carrier may beprepared. Methods for preparation of these compositions are known tothose skilled in the art. The contemplated compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 75-85%.

Kits

Also provided herein are kits. Typically, a kit includes a carrierpeptide, as described previously. In some embodiments, a kit includes acarrier peptide and a biologically active molecule and/or imaging agent.In certain embodiments, a kit can include one or more delivery systems,e.g., for a biologically active molecule, imaging agent, carrierpeptide, or any combination thereof, and directions for use of the kit(e.g., instructions for administering to a subject). In certainembodiments, a kit can include a biologically active molecule and/or animaging agent, a carrier peptide, and/or a complex of a biologicallyactive molecule or imaging agent and a carrier peptide. In someembodiments, the kit can include a carrier peptide and a label thatindicates that the contents are to be administered to a subject with abiologically active molecule or imaging agent.

EXAMPLES General Materials:

All mice used (B6SJLF1) were female and were purchased from the JacksonLaboratories. Mice were maintained and used following an IRB-approvedprotocol. Bacterial β-galactosidase was purchased from Calbiochem(Catalog #345788). Human IgG and IgM were purchased from Sigma (ProductNumbers I 4506 and I8260 respectively). The 4G8 monclonal antibody (cat#SIG-39220) was purchased from Covance (Emeryville, Calif.). LDL receptorantibody was from abcam (Cat # ab30532). All peptides were synthesizedat the Mayo Proteomic Core Facility.

Preparation of peptide-protein complex for delivery in the brain:Required amount of the peptide and protein were mixed in a final volumeof 300 μL PBS (phosphate buffered saline), and incubated at roomtemperature for 60 minutes. The mixture was vortexed for a few seconds,every fifteen minutes during the incubation period.

The mixture was injected intravenously as a bolus into the lumen of thefemoral vein. This was accomplished using a heat pulled PE50 catheter.At the conclusion of the experiment, the mouse was euthanized with alethal dose of sodium pentobarbital. Each mouse was perfused with 10 mLPBS. This perfusion was accomplished through the standard trans cardialmethod. The brain was removed from the skull and positioned to make aninitial coronal slice at −2.0 mm bregma. Subsequently, 25 μm coronalsections were cut on a cryostat and placed on charged slides forstaining of β-galactosidase activity.

Staining for beta-galactosidase enzyme activity: Evaluation ofβ-galactosidase enzymatic activity was accomplished by an initial 15minute fixation of the brain sections in 0.25% glutaraldehyde. Theslides were washed with 3 changes of PBS for 5 minutes each and thenrinsed in distilled water for 5 minutes. The brain sections wereincubated in X-Gal (0.2%) working solution, pH 7.38, for 18 hours at 37°C. in covered containers. Following this incubation the sections weredehydrated and coverslips were applied.Imaging by microSPECT: Micro SPECT/CT experiments were conducted on aGamma Medica X SPECT System (GE Healthcare). Human IgG (Sigma) and IgM(Sigma) were labeled to a high specific activity using the Chloramine-Tmethod. 80 μg (500 μCi) of each immunoglobulin (corresponds to 0.53nanomole of IgG and 0.13 nanomole of IgM assuming molecular weights of150 Kd and 600 Kd for IgG and IgM, respectively) was mixed with 70-foldmolar excess of K16ApoE (as required for experiment) for 1 hour at roomtemperature and was administered in each mouse through the use of acatheter in the femoral vein. Immediately subsequent to the intravenousbolus injection, the mice were imaged every hour for a total of 6 hours.At the completion of the 6 hour time point, each mouse was euthanizedand the systemic blood supply was transcardially perfused with 10 mLphosphate buffered saline, and imaged after 30 minutes.

Example 1 Delivery of Radiolabeled IgG Across the BBB

The delivery of radiolabeled IgG across the BBB in different brainregions in mice with and without the peptide K₁₆ApoE (SEQ ID NO:22) wasevaluated. In addition, the integrity of the BBB after delivery of IgGwith K₁₆ApoE was examined. 3 μmol of ¹²⁵I-IgG were mixed with varyingamounts of the peptide K₁₆ApoE (0-55 μmol), and the mixture injectedintravenously into the mice. ¹³¹I-IgG not complexed with the peptide wasinjected into the same animals at 59 minutes and allowed to circulatefor 1 minute subsequent to the initial femoral vein injection of¹²⁵I-IgG+K₁₆ApoE. Uptake of ¹²⁵I-IgG and ¹³¹I-IgG were measured indifferent areas of the brain after 60 minutes.

TABLE 1 +ApoE, +ApoE, +ApoE, +ApoE, IgG, 3 μmol 8 μmol 16 μmol 32 μmol55 μmol PS × 10⁶ (ml/g/s) Cortex 0.13 +− 0.02 0.30 0.47 9.58 15.30Caudate 0.09 +− 0.01 0.35 0.86 16.65 22.60 Hippo 0.17 +− 0.02 0.58 1.0512.52 20.90 Thalamus 0.13 +− 0.02 0.29 1.37 19.60 21.00 Brainstem 0.20+− 0.02 0.45 1.64 22.90 18.40 Cerebellum 0.15 +− 0.02 0.48 0.72 13.4321.60 Vp (ul/g) Cortex 13.53 +− 0.98  21.39 21.22 16.41 25.59 Caudate8.53 +− 0.52 14.44 11.67 12.89 20.10 Hippo 15.13 +− 2.35  17.49 16.9512.67 21.83 Thalamus 13.79 +− 1.09  19.96 21.00 18.34 26.92 Brainstem22.32 +− 1.51  19.02 31.50 20.54 34.60 Cerebellum 20.79 +− 1.24  21.2823.74 19.79 24.31 N (no of mice) 4 2 2 2 1PS values reflect the amount of ¹²⁵I-IgG that has crossed the BBB,whereas Vp values indicate the amount of ¹³¹I-IgG at the BBB but whichhas not crossed the BBB.

As shown in Table 1, the PS values with IgG alone are very small for allthe brain regions, indicating very little transport of the IgG acrossthe BBB. However, the values increased significantly (˜100-fold at 55μmol of K₁₆ApoE) with increasing amounts of the delivery peptide.

On the other hand, the Vp values remained virtually unchanged in all thebrain regions examined, indicating ¹³¹I-IgG failed to cross the BBB(since it was not complexed with the peptide), and attesting to thenotion that the BBB was not damaged due to prior exposure to the peptide(during delivery of ¹²⁵I-IgG complexed with K₁₆ApoE).

Example 2 Delivery of Alexa-IgG Assisted by K₁₆ApoE into Brain

In this experiment, AlexaIgG488 was injected into mice with or withoutmixing with K₁₆ApoE peptide (SEQ ID NO:22). Brain sections were made 1hour after delivery. As shown in FIG. 1, the complexed agent showssignificantly brighter contrast and enhancement of brain structurescompared to the uncomplexed AlexaIgG488.

Example 3 Delivery of ApoE Peptide (without K₁₆ Moiety) Across the BBBand Binding of Peptide to LDL Receptors

The experiments will evaluate the ApoE peptide (without the K₁₆ moiety)crossing of the BBB and whether it will bind/localize to LDL receptors(LDLR) expressed in the brain. An FITC-labeled ApoE peptide will besynthesized and injected into mice. Brain specimens will be collectedafter 1 hour following injection, and slides will be prepared with brainsections of 10 microns thickness. The slides will be stained withantibody against LDLR and evaluated to determine whether the signals forLDLR and the ApoE peptide are co-localized.

Example 4 Location of K₁₆ApoE Following Delivery Across the BBB

The experiments will determine the location of protein delivery by thecarrier peptide (K₁₆ApoE) within the brain. K₁₆ApoE (e.g., SEQ ID NO:22)+Alexa-IgG (green fluorescence) conjugate will be prepared by mixingsolutions of each at the desired concentrations and incubating themixture at room temp for 30 minutes. The complex will be injected intomice and brain specimens collected after 1 hour. Slides will be preparedas above, and immunostained for GFAP, calbindin and neuregulin. Ifco-localization of IgG and GFAP is seen, such a result may indicate thatthe delivered IgG reached the astrocytes. Co-localization of IgG andcalbindin or neuregulin, however, may indicate that the delivered IgGreached the neurons.

Example 5 Compromised Integrity of the BBB by K₁₆ApoE

These experiments will evaluate whether the K₁₆ApoE (e.g., SEQ IDNO:22), alone or following formation of a complex, compromises theintegrity of the BBB. A complex with K₁₆ApoE+AlexaRedIgG will beprepared and injected into mice. After 1 hour uncomplexed AlexaGreenIgGwill be injected. Brain specimens will be collected 1 hour after thesecond injection, and slides will be prepared using 10 micron sections.The sections will be visualized using confocal microscopy. In othermice, the reverse the experimental approach will be implemented usingAlexaGreenIgG complexed with K₁₆ApoE and uncomplexed AlexaRedIgG. If thepeptide does not compromise the BBB, then only red fluorescence will beseen in cells in the brain. However, if the peptide does compromise theBBB, then both red and green fluorescence will be seen, and vice versa.

Example 6 Evaluation of microSPECT

A microSPECT machine can image and quantify radioactivity in varioustissues in live animals under anesthesia. If K₁₆ApoE indeed carries aprotein (radioactive) across the BBB, then there should be moreradioactivity in the brain of a mice injected with such apeptide-protein complex than in the brain of a mice injected with theradioactive protein alone.

IgG 4.1 (an antibody against the amyloid precursor protein (APP)) wasradioiodinated. IgG4.1 alone or previously complexed with K₁₆ApoE (SEQID NO:22) was injected in a number of mice. The mice were anesthesizedand the brain was imaged with a microSPECT machine at 1 hour intervals.Perfusion was done for some mice after six hours of quantitative imagingfollowed by imaging after 30 minutes.

The radioactivity measured at each time point for each mice was plottedagainst time (FIG. 2). Seven animals were used in total, they arenumbered M3-M9. M3 and M4 mice were not perfused. Two mice (M5 and M7)receiving IgG 4.1 were perfused after 6 hours of imaging, and three mice(M6, M8 and M9) receiving the IgG complexed with the delivery peptidewere perfused after 6 hours of imaging followed by imaging at 6.5 hours.

It is clear from the data presented in FIG. 2 that ˜2-fold moreradioactivity was registered in the brains of mice delivered with theIgG complexed with the peptide than the IgG alone. This differenceincreased to nearly 5-fold when the animals were perfused.

Radioactivity in the brains of mice receiving IgG alone indicated IgGmolecules were retained in the capillaries. Increased accumulation ofradioactivity in the brains of mice receiving the IgG complexed with thedelivery peptide indicated IgGs were present in the capillaries as wellas having crossed the BBB and retained in the brain. If the IgG crossesthe BBB and is retained in brain cells, then perfusion of the brainsshould eliminate IgGs in the capillaries but IgGs that have crossed theBBB should still be retained, which is essentially seen for mice M6, M8and M9, providing strong indication that the peptide helps carry theprotein (IgG) through the BBB into the brain.

Example 7 Delivery of a Protein Across the BBB

To evaluate the potential of K16ApoE (SEQ ID NO: 22) for delivering aprotein across the BBB, the enzyme β-galactosidase was injected in micewith or without prior mixing with K16ApoE (at a protein to peptide molarratio of 1:70). The experiment was repeated more than a dozen times. Inthis series of experiments, intense β-galactosidase activity wasobserved in mice brain when the enzyme-peptide mix was injected andbrain slices were prepared for enzyme activity staining 6 hours afterinjection, whereas no activity was seen when the enzyme was injectedalone (FIG. 3A). High-magnification scans of the images ofβ-galactosidase-stained sections in the brain showed that the enzyme wasdelivered in virtually every area of the brain, and stained bothastrocytes and neurons, however, pyramidal cells in the hippocampusshowed much weaker staining for β-galactosidase activity compared tocells in other areas of the brain. The weak β-galactosidase activitystaining in the pyramidal cells does not appear to be due to lowexpression of LDLR in mouse hippocampus since cells in this regiondisplay strong immunohistochemical staining for the receptor. It isspeculated that either X-gal or 5-bromo-4-chloroindole or both somehowleach out of this region faster than other regions of the brainresulting to a faint signal for β-galactosidase activity.

To assess if the delivery of β-galactosidase in the brain via K16ApoE isLDLR-mediated, β-galactosidase staining in liver, brain, kidney, heartand lung after injection of the enzyme with and without mixing withK16ApoE (SEQ ID NO: 22) was also evaluated. The most intense stainingwas observed in liver and brain, followed by staining in lung, heart andkidney (FIG. 3B-G). This pattern of uptake of beta-galactosidase byK16ApoE in various organs approximately follows the pattern of reportedLDLR expression pattern suggesting that delivery of β-galactosidasethrough K16ApoE is LDLR-mediated.

Example 8 Delivery of IgG and IgM Using K16ApoE

Since many potentially therapeutic proteins against brain-associateddisorders are immunoglobulins, delivery of IgG and IgM through K16ApoEin mouse brain was evaluated by micro-single photon emission computedtomography (microSPECT) imaging. For this, radioiodinated human IgG(Sigma, product # I 4506) and human IgM (Sigma, product # I 8260) weremixed with K16ApoE (SEQ ID NO: 22) and injected intravenously.Anesthetized animals were then subjected to imaging for 6 hours at 1hour interval. Cardiac perfusion was then performed, after which finalimaging was done at 30 minutes post-perfusion. Results from theseexperiments provide a number of indications (FIG. 4): First, it appearsthat peak delivery of both IgG and IgM into brain happens around 2 hourspost-injection of the peptide-protein mix, after which uptake of theproteins in the brain remains relatively unchanged up to 6 hours(maximum time point tested). Second, cardiac perfusion appears to removemost of the radioactive protein in the vasculature, concomitantlyincreasing the difference between apparent brain-uptake of the proteinwith and without the carrier peptide (˜5-fold). Finally, the resultssuggest that about 1% of the injected immunoglobulins are transportedinto the brain by K16ApoE under the experimental condition. It isinteresting to note that the extent of delivery of the radiolabeled IgGand IgM into kidney, liver/spleen and heart remained virtually unchangedirrespective of whether the proteins were delivered with or without thecarrier peptide. Cardiac perfusion also did not change the accumulationof radioactivity in these organs, implying that transport of proteins inthese organs does not depend upon crossing of a biological barrier.

Example 9 Efficacy of Delivered Therapeutic

For its activity, β-galactosidase does not need to bind with anymolecular entity in the brain. However, for all therapeutic purposes,the delivered molecule in the brain must be able to recognize and bindwith its cognate target molecule. To test this premise, an antibodyagainst amyloid beta peptide (4G8 from Cavance) was delivered viaK16ApoE in the brains of mice which model for Alzheimer's disease. Thisantibody is known to recognize amyloid beta plaques. The resultsindicate that the antibody delivered in this manner labeled the amyloidplaques in the brain of these mice as well as identified the plaqueswith the antibody through standard immunohistochemistry (FIG. 5).Similar results were also obtained with another antibody (4.1 IgG, 32)known to label these plaques.

Example 10 Transportation of Protein without Damage to BBB

A transporter that efficiently carries a protein in the brain should notdo so by impairing the integrity of the BBB. To evaluate if K16ApoE (SEQID NO: 22) impairs the integrity of the BBB, the peptide was firstinjected intravenously, then injected β-galactosidase at different timeintervals. The control mice received the enzyme mixed with the peptide.Mice were perfused and sacrificed 6 hours after β-galactosidaseinjection, brain slices were made and prepared for β-galactosidasestaining Results presented in FIG. 6 show increasingly weakerβ-galactosidase activity in the brains of mice receiving β-galactosidaseat 1, 5 and 10 minutes after injection of K16ApoE and no visiblestaining thereafter. These results indicate that most of the injectedK16ApoE becomes bound with proteins and cells in the circulation, andthis binding becomes virtually complete within 10 minutes after which nofree K16APoE remains in circulation. Free K16ApoE that remains at earlytime points becomes bound with beta-galactosidase and carries the enzymein the brain. The peptide by itself and/or being bound with bloodproteins/cells did not seem to affect the BBB as no β-galactosidaseenzyme activity was seen in the brain from 10 min to 4 hours.

Example 11 Necessity of Covalent Linking of Hydrophillic Amino AcidChain and Carrier Peptide

The necessity of covalent linking of the K16 and ApoE (SEQ ID NO: 13)peptide moieties was evaluated for non-covalent transport of a proteinacross the BBB. For this, β-galactosidase was mixed with either the ApoEpeptide, the peptide K16, ApoE plus K16 or with K16ApoE, and injected inmice. Brain sections from these mice show β-galactosidase staining onlyin slides from mice receiving β-galactosidase mixed with K16ApoE,indicating that linking of the K16 and the ApoE peptide moieties iscritical for transport of a protein in a non-covalent manner (FIG. 7).

Example 12 Testing Mutant ApoE Peptides for their Potential asNon-Covalent Transporters of Proteins Across the BBB

β-galactosidase was mixed with equal amounts of various peptidetransporters and injected into mice. Slides were prepared using 25 μmsections of the mice brains one hour after injection followed bydevelopment of enzyme staining (FIG. 8). The following peptides wereevaluated:

(SEQ ID NO: 126) A: KKKK KKKK KKKK KKKK LRLR LANH LRKL RKRL LRDA;(SEQ ID NO: 127) B: KKKK KKKK KKKK KKKK LRLR LASH LRKL RKRL LRDL;(SEQ ID NO: 128) C: KKKK KKKK KKKK KKKK LRVR LANH LRKL RKRL LRDL.As shown in FIG. 8, mutant A was worse than the wild-type ApoEblood-brain barrier agent, while mutants B and C were able to transportthe β-galactosidase at least as well as wild-type or better.

Example 13 Time Course for β-Galactosidase Delivery in Mouse Brain withK16ApoE

In each mouse, 1.38 nanomoles of β-galactosidase was mixed with 89nanomoles of K16ApoE (SEQ ID NO: 22) and injected intravenously. 25 μmbrain slices were prepared for staining for β-galactosidase activity atindicated time points (FIG. 9).

FIG. 8 provides the following time points:

A—Beta-galactosidase, no peptide, 6 h.B—Beta-galactosidase+peptide, 1 h.C—Beta-galactosidase+peptide, 2 h.D—Beta-galactosidase+peptide, 5 h.E—Beta-galactosidase+peptide, 10 h.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A carrier peptide comprising the sequence:X_(n)-[B]_(m) or a pharmaceutically acceptable salt thereof, wherein: Xis a hydrophilic amino acid; B is a blood-brain barrier agent; n is aninteger from 4 to 50; and m is integer from 1 to 3; wherein theblood-brain barrier agent is not L-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R(SEQ ID NO: 141).
 2. The carrier peptide of claim 1, wherein each X isindependently chosen from arginine, asparagine, aspartic acid, glutamicacid, glutamine, histidine, lysine, serine, threonine, and tyrosine. 3.The carrier peptide of claim 1, wherein X is lysine.
 4. The carrierpeptide of claim 1, wherein n is chosen from 4, 8, 12, 16, and
 20. 5.The carrier peptide of claim 1, wherein n is
 16. 6. The carrier peptideof claim 1, wherein m is
 1. 7. The carrier peptide of claim 1, wherein Bis a receptor binding domain of an apolipoprotein.
 8. The carrierpeptide of claim 7, wherein the a receptor binding domain of anapolipoprotein is chosen from the receptor binding domain of ApoA, ApoB,ApoC, ApoD, ApoE, ApoE2, ApoE3, and ApoE4.
 9. The carrier peptide ofclaim 8, wherein the receptor binding domain of an apolipoprotein ischosen from the receptor binding domain of ApoB and ApoE.
 10. Thecarrier peptide of claim 1, wherein the blood-brain barrier agentcomprises a polypeptide sequence having at least 80% sequence identityto: (SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A.


11. The carrier peptide of claim 1, wherein the blood-brain barrieragent comprises a polypeptide sequence having at least 80% sequenceidentity to: (SEQ ID NO: 14)S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H.


12. The carrier peptide of claim 1, wherein the blood-brain barrieragent comprises a polypeptide sequence having at least 80% sequenceidentity to: (SEQ ID NO: 15)Y-P-A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y-L-N-L-V-T-R-Q-R-Y*; (SEQ ID NO: 16)A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R- H-Y-L-N-L-V-T-R-Q-R-Y*;(SEQ ID NO: 17) Y-P-S-D-P-D-N-P-G-E-D-A-P-A-E-D-L-A-R-Y-Y-S-A-L-R-H-Y-I-N-L-I-T-R-Q-R-Y*; or (SEQ ID NO: 18)A-P-L-E-P-V-Y-P-G-D-D-A-T-P-E-Q-M-A-Q-Y-A-A-E-L-R-R-Y-I-N-M-L-T-R-P-R-Y*,

wherein Y* is tyrosine or a tyrosine derivative.
 13. The carrier peptideof any one of claims 10-12, wherein the polypeptide is less than 100amino acids in length.
 14. The carrier peptide of claim 1, wherein thecarrier peptide is chosen from: (SEQ ID NO: 19)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 20)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 21) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 22)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 24)K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 25)K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 26)K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V- E-G-S-H;(SEQ ID NO: 27) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S- N-K-F-V-E-G-S-H; and(SEQ ID NO: 28) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H.


15. A complex, or a pharmaceutically acceptable salt thereof, comprisinga biologically active molecule or an imaging agent associated with acarrier peptide, wherein the carrier peptide comprises the sequence:X_(n)-[B]_(m) wherein: X is a hydrophilic amino acid; B is a blood-brainbarrier agent; n is an integer from 4 to 50; and m is integer from 1 to3, wherein the blood-brain barrier agent is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R (SEQ ID NO: 141). 16.-28.(canceled)
 29. The complex of claim 15, wherein the imaging agent ischosen from: ¹²⁵I-IgG and magnevist.
 30. The complex of claim 15,wherein the biologically active molecule is chosen from a: polypeptide;oligonucleotide; plasmid; small molecule; antibody; antibody fragment;carbohydrate; polysaccharide; lipid; glycolipid; antigen; and antigenicpeptide.
 31. The complex of claim 30, wherein the biologically activemolecule is chosen from a: polypeptide; oligonucleotide; and plasmid.32. The complex of claim 30, wherein the oligonucleotide is chosen froma: coding DNA sequence; antisense DNA sequence; mRNA, antisense RNAsequence; RNAi; and siRNA.
 33. The complex of claim 30, wherein thesmall molecule is a therapeutic agent.
 34. The complex of claim 15,wherein the biologically active molecule or imaging agent isnon-covalently bound to the carrier peptide.
 35. A method oftransporting a biologically active molecule or imaging agent across theblood-brain barrier of a subject, the method comprising administering tothe subject a complex, or a pharmaceutically acceptable salt thereof,comprising the biologically active molecule or imaging agent associatedwith a carrier peptide; wherein the carrier peptide comprises thesequence:X_(n)-[B]_(m) or a pharmaceutically acceptable salt thereof, wherein: Xis a hydrophilic amino acid; B is a blood-brain barrier agent; n is aninteger from 4 to 50; and m is integer from 1 to 3, wherein theblood-brain barrier agent is not L-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R(SEQ ID NO: 141).
 36. A method of treating a brain disorder in asubject, the method comprising administering to the subject a complex,or a pharmaceutically acceptable salt thereof, comprising a biologicallyactive agent associated with a carrier peptide, wherein the carrierpeptide comprises the sequence:X_(n)-[B]_(m) wherein: X is a hydrophilic amino acid; B is a blood-brainbarrier agent; n is an integer from 4 to 50; and m is integer from 1 to3, wherein the blood-brain barrier agent is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R (SEQ ID NO: 141).
 37. The methodof claim 36, wherein the brain disorder is chosen from: meningitis,epilepsy, multiple sclerosis, neuromyelitis optica, late-stageneurological trypanosomiasis, Parkinson's, progressive multifocalleukoencephalopathy, De Vivo disease, Alzheimer's disease, HIVEncephalitis, and cancer.
 38. A method of imaging the central nervoussystem of a subject, the method comprising administering to the subjecta complex, or a pharmaceutically acceptable salt thereof, comprising animaging agent associated with a carrier peptide, wherein the carrierpeptide comprises the sequence:X_(n)-[B]_(m) wherein: X is a hydrophilic amino acid; B is a blood-brainbarrier agent; n is an integer from 4 to 50; and m is integer from 1 to3; and imaging the central nervous system of the subject, wherein theblood-brain barrier agent is not L-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R(SEQ ID NO: 141).
 39. A pharmaceutical composition comprising a complex,or a pharmaceutically acceptable salt thereof, comprising a biologicallyactive molecule or imaging agent, a carrier peptide, and apharmaceutically acceptable carrier; wherein the carrier peptidecomprises the sequence:X_(n)-[B]_(m) wherein: X is a hydrophilic amino acid; B is a blood-brainbarrier agent; n is an integer from 4 to 50; and m is integer from 1 to3, wherein the blood-brain barrier agent is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R (SEQ ID NO: 141). 40.-42.(canceled)
 42. A blood-brain barrier agent comprising the sequence:(SEQ ID NO: 29) L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8-R-D-X9

wherein: X1 is selected from the group consisting of A, L, S, and V; X2is selected from the group consisting of L and M; X3 is selected fromthe group consisting of A and S; X4 is selected from the groupconsisting of N, S, and T; X5 is selected from the group consisting of Kand N; X6 is selected from the group consisting of L, M, and V; X7 isselected from the group consisting of R and P; X8 is selected from thegroup consisting of L and M; and X9 is selected from the groupconsisting of A and L; wherein the carrier peptide does not comprise:(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A.


43. The blood-brain barrier agent of claim 42, wherein the blood-brainbarrier agent is selected from the group consisting of: (SEQ ID NO: 30)L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 31)L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 32)L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 33)L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 34)L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 35)L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 36)L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 37)L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 38)L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 39)L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 40)L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 41)L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 42)L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 43)L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 44)L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 45)L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 129)L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; and (SEQ ID NO: 130)L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.

44.-49. (canceled)
 50. A complex, or a pharmaceutically acceptable saltthereof, comprising a biologically active molecule or an imaging agentassociated with a carrier peptide, wherein the carrier peptide comprisesthe sequence: (SEQ ID NO: 143)[X]n-L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8- R-D-X9

wherein: X is a hydrophilic amino acid; n is an integer from 4 to 50; X1is selected from the group consisting of A, L, S, and V; X2 is selectedfrom the group consisting of L and M; X3 is selected from the groupconsisting of A and S; X4 is selected from the group consisting of N, S,and T; X5 is selected from the group consisting of K and N; X6 isselected from the group consisting of L, M, and V; X7 is selected fromthe group consisting of R and P; X8 is selected from the groupconsisting of L and M; and X9 is selected from the group consisting of Aand L.
 51. The complex of claim 50, wherein the carrier peptide isselected form the group consisting of: (SEQ ID NO: 19)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 20)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A; (SEQD NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L- R-K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 46)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 47)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 48) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 49)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 50)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 51)K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 52)K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 53) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 54)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 55)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 56)K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 57)K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 58) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 59)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 60)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 61)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 62)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L- L-R-D-A;(SEQ ID NO: 63) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 64)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 65)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 66)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 67)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L- M-R-D-A;(SEQ ID NO: 68) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 69)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 70)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 71)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 72)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 73) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 74)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 75)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 76)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 77)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L- L-R-D-A;(SEQ ID NO: 78) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 79)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 80)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 81)K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 82)K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 83) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 84)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 85)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 86)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 87)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L- L-R-D-A;(SEQ ID NO: 88) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 89)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 90)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 91)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 92)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L- L-R-D-A;(SEQ ID NO: 93) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 94)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 95)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 96)K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 97)K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 98) K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 99)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 100)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 101)K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 102)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L- M-R-D-A;(SEQ ID NO: 103) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 104)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 105)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 106)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 107)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L- M-R-D-A;(SEQ ID NO: 108) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 109)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 110)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 111)K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 112)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L- L-R-D-A;(SEQ ID NO: 113) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 114)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 115)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 116)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 117)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L- M-R-D-A;(SEQ ID NO: 118) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 119)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 120)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 121)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 122)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L- L-R-D-A;(SEQ ID NO: 123) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 124)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 125)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 131)K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 132)K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L- L-R-D-L;(SEQ ID NO: 133) K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 134)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 135)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 136)K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 137)K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L- L-R-D-L;(SEQ ID NO: 138) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 139)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 140)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.